α-β C4BP-type recombinant heteromultimeric proteins

ABSTRACT

A recombinant heteromultimeric protein including at least (a) a polypeptide fusion molecule A consisting of a C4BP α-chain C-terminal fragment and a polypeptide fragment heterologous to said α chain, and (b) a polypeptide fusion molecule B consisting of a C4BP β-chain C-terminal fragment and a polypeptide fragment heterologous to said β-chain, wherein (a) and (b) are linked in the C-terminal portion to form said multimeric protein.

This application is a Continuation Application of U.S. Ser. No.08/983,474, filed on Jun. 30, 1998, which has issued as U.S. Pat. No.7,074,411. That application is incorporated by reference herein in itsentirety.

The present invention relate to heteromultimeric fusion proteins of theC4BP type, to compositions which comprise them and to the process forpreparing them. More specifically, this invention relates toheteromultimeric fusion proteins which are derived by combining α and βmonomer of the C4BP protein, or fragments of these monomers, with thesemonomers being fused to polypeptides which are derived from functionallyactive proteins having ligand or receptor properties.

The numbers in brackets relate to the bibliographical list at the end ofthe text.

“C4BP-binding protein” (C4BP), previously termed proline-rich protein,is an important protein both in the coagulation system (1) and in thecomplement system (2, 3). The major form of C4BP consists of 7 identicalα chains of 75 Kd and of one βchain of 45 Kd. The nucleotide sequence ofthe cDNA and the protein sequence of the α chain have been determined(4). A complete description of human C4BP, its isolation and itscharacterization have been given in (2); an update of knowledgeregarding this molecule has been presented in summary form in (5).

Up until 1990, it was not known that the two chains, α and β, existed;it was only in 1990 that A. Hillarp demonstrated for the first time thata new subunit, which was designated the β chain, and which contains thesite for binding to the S protein (6, 7), existed in the multimericprotein.

The BIOGEN Patent Application WO 91/1146 describes multimeric proteinsof the C4BP type which consist solely of α monomers in which theN-terminal moiety has been replaced by a fragment of the CD4 protein.

However, the constructs which are described in this document only makeuse of the α chain, since the β chain was unknown at that time. Thedrawback of these constructs is that it is extremely difficult, on theone hand, to control the physical state of the synthetic molecule, thatis the number of monomers which combine with each other, and, on theother hand, it is extremely difficult, when it is desired to combine twodifferent functional moieties within the multimer, to control theproportions of these functional elements.

Therapeutic agents which function at the level of the immune system,whether the agent be a cellular or humoral agent, or animmunointervention agent, have developed in a large number ofdirections; however, the areas of application of therapeuticimmunointerventions using antibodies, in particular monoclonalantibodies, currently remain modest due to the act that the phenomenawhich take place after the antibodies have bound to the cell are poorlycontrolled. Thus, it is currently only known how to use theseantibodies, even when humanized, for the purposes of cell destruction.

The development of bispecific antibodies has also been envisaged, withsome of these antibodies comprising one moiety which is capable ofbinding to an antigen, with the other moiety having the role of a ligandin relation to a receptor, thereby making it possible to direct theantibody plus antigen toward a cell system (8). However, these systemsdo not enable several ligands to be combined in one and the samecomplex, which appears in some cases to be a prerequisite for triggeringan immune response.

Another system which has been proposed for achieving multimers is basedon the IgM Fc; this system suffers from several drawbacks: the maindrawback is that of creating molecular species of varying size, leavingfree sulfhydryl residues which are able to react with plasma moleculesor cell surfaces. Furthermore, the cell receptor-binding andcomplement-activating functions of the Fc fragment may be undesirable.

By contrast, a recombinant heteromultimeric molecule can make itpossible to combine several antibody functions, or several enzymemolecules or several antigens, or fragments or mixtures thereof, therebycreating a multivalent tracer which possesses a potential for amplifyingthe detected signal which is superior to that of a fusion protein whichcombines a single antibody or a bispecific antibody and an enzyme orantigen molecule.

This approach makes it possible to envisage, in addition to opsonizationby means of triggering cellular immunity, the induced implementation ofhumoral immunity by means of activating complement.

The object of the present invention is therefore to develop recombinantsoluble heteromultimeric chimeric molecules which combine differentfunctions in one and the same molecule, with a view to achievingimmunointervention in human immune pathologies. These molecules willmake it possible to intervene in the physiopathological mechanisms ofdifferent ailments, in particular in the spheres relating to:

-   -   the pathology of the transport and elimination of immune        complexes by the erthyrocytes, with an application, in        particular, to disseminated lupus erythematosus, or to HIV        infections,    -   the capture of antigens which is mediated by Fc receptors on the        surface of the cells of the monocyte/macrophage cell line,    -   modulation by molecules having soluble CD16 activity,    -   the prevention of anti-erythrocyte Rh(D) alloimmunization,    -   the inhibition of cell penetration by the HIV virus using        soluble forms of CD4, antibodies which are directed against the        constituents of the virus, and/or molecules having enzymic        functions.

The present invention relates to a recombinant multimeric protein whichis characterized in that it comprises at least:

a) a polypeptide fusion molecule A, which consists of a C-terminalfragment of the α chain of C4BP, contained between amino acids 549 and124)SEQ ID NO: 11), and a polypeptide fragment which is heterologous inrelation to said α chain,

b) a polypeptide fusion molecule B, which consists of a C-terminalfragment of the β chain of C4BP, contained between amino acids 235 and120 (SEQ ID NO: 12), and a polypeptide fragment which is heterologous inrelation to the β chain,

Preferably, a recombinant multimeric protein according to the inventionis characterized in that the C-terminal fragment of the α chain iscontained between amino acids 549 and 493, and in that the C-terminalfragment of the β chain is contained between amino acids 235 and 176.The fusion molecules are reassociated by forming disulfide bridgesbetween the cysteines in positions 498 and 510 of the C-terminal end ofthe α chain and the cysteines in positions 199 and 185 of the C-terminalend of the β chain.

Any chimera between the α and β chains which links, where appropriate, acysteine of the α chain and a cysteine of the β chain in the A or Bfusion molecule, or in the two fusion molecules, is also included in thescope of the constructs of the multimeric proteins of the invention.

In particular, the possibility of altering the spacing between the twocysteines can enable the number of monomers which are included in theconstitution of the multimer to be altered.

By way of illustration, an increase in the distance between thecysteines can lead to an increase in the number of monomers which areincluded in the multimer; on the other hand, a decrease in this distancewould result in a decrease in this number. In certain cases, it can beadvantageous to alter this distance so as to alter the controlledreassociation of the two types of chain carrying a ligand or a receptor,both with regard to the number of chains and their proportions.

In the present invention, a multimerizing system has been developedwhich makes it possible to obtain heptameric and octameric formulaeusing the C-terminal ends of the basic chains of the C4BP molecule.Multimerization of molecules whose C-terminal ends have been replaced bythe C4BP α C-terminal moiety or the C4BP β C-terminal moiety produces anoctameric form.

Whatever the case, the fragment which is derived from the β chain ofC4BP should lack the sites for attachment to the S protein, which sitesare located in the two SCRs of the proximal moiety of the N-terminal endof the β chain.

Thus, a recombinant multimeric protein according to the invention ischaracterized in that the ratio of the number of monomers α/β variesbetween 7/1 and 5/3 and is preferably 7/1 when the fragments of theC-terminal moieties have a homogeneous origin in fragments A and Babove.

Ideally, the multimeric recombinant molecule is a protein which onlycombines human constituents, thereby avoiding any xenogenic immunizationand not activating complement apart from specific functions which areintentionally added in this sense. Such a molecule should not interactwith cell receptors or plasma molecules, and should thereby ensure thatthe activity of the chimeric molecule is improved due to itsmultivalence and that the life span of the molecule is extended.

A multimeric protein according to the present invention exhibitsproperties relating to immuno-intervention, in particular an ability tomodulate the activity of complement with the aim of generatingopsonization artificially. This result is obtained as a consequence ofthe heterofunctional character which can be attributed to this proteinas a result of the contribution made by the heterologous N-terminalfragments.

To this end, a recombinant protein according to the invention ischaracterized in that the heterologous fragments in A and in B arederived from specific ligands of the immune system, in particular fromlymphocyte surface proteins of the CD type, from antibodies or antibodyfragments, or from antigens or antigen fragments; the heterologousfragments in A and B can be selected, depending on the application whichis desired for the recombinant protein, and without this list beingexhaustive, from the following polypeptides having a ligand activity:

-   i)—fragments derived from lymphocyte proteins are CD4, CD8, CD16 and    CD35 (or CR1),-   ii)—antibodies or antibody fragments having an anti-erythrocyte    specificity, in particular an anti-Rh(D) specificity,-   iii)—antigens, in particular vaccinating antigens such as the pre-S2    of hepatitis B virus,-   iv)—an enzyme which is intended for therapeutic purposes or, more    specifically, the fragment of this enzyme which corresponds to the    active site of said enzyme, which fragment can be fused to the    C-terminal moiety of the alpha chain in order to form the A    fragment, with it then being possible for the B fragment to consist    of any type of polypeptide such as cited in i) to iii).

Even more particularly, combinations of A and B which are particularlyadvantageous for implementing the immunointervention of the inventioncan be multimeric proteins in which the polypeptide fusion fragmentscontain:

-   v)—in A, CD4 or a derivative of CD4, and    -   in B, the sc Fv of an anti-Rh(D) antibody, or of other        antibodies, in particular neutralizing antibodies, or antigen        targets.

Another advantageous construct is a multimeric protein in which thepolypeptide fusion fragments contain:

-   -   in A, an antigen, in particular a vaccinating antigen or a        therapeutic enzyme or a CD35 (or CR1) or an antibody, or any        fragment thereof which possesses the ligand property of the        whole molecule;    -   in B, an antibody or a fragment thereof which has retained its        paratope;

Another advantageous recombinant multimeric proteins are those in whichthe polypeptide fusion fragments contains:

-   -   in A, a vaccinating immunogen, and    -   in B, a CD4 or a derived molecule, provided that it retains the        ligand property of the whole molecule.

In addition, the present invention is directed toward eukaryotic orprokaryotic cells which are transduced with one or more plasmidscontaining a heterologous nucleic acid sequence and encoding at leastone polypeptide fusion molecule A and one polypeptide fusion molecule B.

The different cell lines which can be used for being transduced withplasmids are preferably eukaryotic cells which are capable of carryingout post-translational glycosylation; those which may be mentioned byway of example are yeasts or animal cell lines such as cells of thefibroblast type, such as BHK or CHO cells, or else lymphocyte cell linessuch as immortalized lymphocytes.

According to different embodiments of the present invention, the cellscan be:

-   -   cotransduced with two separate plasmids, or    -   transduced with a plasmid encoding a first polypeptide and then        supertransduced with the second plasmid encoding the second        polypeptide, or    -   result from the fusion of two cells, one of which has been        transduced with the plasmid encoding the first polypeptide while        the other has been transduced with a plasmid encoding the        second-polypeptide.

The cell fusions are carried out using standard methods, either byaction of PEG (polyethylene glycol) or by action of Epstein-Barr virus,or using any other standard method for fusing two different eukaryoticcells.

More specifically, said cells can be transduced with the first plasmid,which is that which was deposited in the C.N.C.M. under No. I-1610 on 12Jul. 1995, and with the second plasmid, which is that which wasdeposited in the C.N.C.M. under No. I-1611 on 12 Jul. 1995.

The present invention also relates to a process for preparing amultimeric protein according to the invention. This process ischaracterized in that it comprises at least the following steps:

-   -   transducing target cell lines with at least one plasmid        containing a heterologous sequence encoding an A chain or a B        chain, as defined above,    -   expressing and isolating of the heterologous A or B chains,    -   placing said polypeptides, in specific proportions, in an        oxidizing medium,    -   isolating the multimers.

Preferably, this preparation process is characterized in that thetransduced cell lines have been either:

-   -   cotransduced with two plasmids carrying DNA sequences which        respectively encode the A and B polypeptides, or    -   transduced with a plasmid encoding a first polypeptide and then        supertransduced with the second plasmid encoding the second        polypeptide, or    -   result from the fusion of two cells, one of which has been        transduced with the plasmid encoding the first polypeptide while        the other has been transduced with a plasmid encoding the second        polypeptide.

Furthermore, the present invention relates to the use of a recombinantprotein as previously defined in the production of a medicament and,more particularly, of a medicament which is intended for:

-   -   prevention of fetomaternal alloimmunization, or    -   the therapy or prophylaxis of viral, bacterial or parasitic        infections,    -   the therapy of autoimmune diseases, in particular disseminated        lupus erythematosus, or alloimmune diseases.

More generally, the present invention relates to the use of arecombinant protein as previously defined in the production of amedicament which makes it possible, depending on the functionality whichis attributed to the ligands or the receptors, to effect animmunointervention, in particular in the opsonization ornon-opsonization of target cells by means of activating, modulating orinhibiting complement.

The skilled person will know, in step with discovering thefunctionalities of certain proteins or of certain ligands or receptors,how to construct a recombinant multimeric protein according to theinvention which is best suited for the sought-after effect.

Advantageously, the use of the multimeric protein according to theinvention is characterized in that it enables complement to besufficiently activated to induce opsonization of cells whose antigenicor epitopic sites are not naturally able to trigger such activation.

A pharmaceutical composition which is characterized in that itcomprises, as the active principle, a recombinant multimeric protein asdescribed above is also included within the scope of the presentinvention. Said pharmaceutical composition can enable the immunotherapyor the immunoprevention of different pathologies, in particular thosewhich are linked to viral or bacterial infections or to autoimmune oralloimmune diseases.

A recombinant protein according to the invention can also be used in adiagnostic test which requires the intervention of at least twodifferent ligands or receptors.

The feasibility of the multimer of very high molecular weight wasverified in a multi-CR1 model (approx. 1.5 million daltons). Thismolecule is functional and inhibits complement activation in a model ofcomplement-dependent, antibody-covered erythrocyte lysis atconcentrations which are lower than those required for monomeric solubleCR1.

The feasibility of heterochimeras which combine different functions wasthen established using, on the one hand, anti-Rh(D) antibody, morespecifically the variable Fv moiety of this antibody, and even morespecifically the single-chain moiety of this variable moiety, termedscFv, which, in the present case, was linked to the CD35 or (CR1)molecule, which is able to inhibit or modulate complement activity; theother system employed is a heteromultimeric system of the CD4/antigentype.

The examples which follow are in no way limiting and only serve todemonstrate the feasibility of the constructs of these recombinantheteromultimers for the purposes of immunointervention; the figureswhich illustrate the examples have the following meanings:

FIG. 1 depicts an expression vector which consists of a plasmid whichcontains the sequence encoding CD4 and which is termed sT4CD4-C4BP;

FIG. 2 depicts a plasmid vector which contains the sequence encodingmultimeric CD16.

FIG. 3 depicts a plasmid vector which contains the sequence encodingmultimeric CR1;

FIG. 4 depicts another plasmid vector, pCDM8, which encodes the samemultimeric CR1;

FIG. 5 depicts a plasmid vector which contains the sequence encoding thescFv of multimeric anti-Rh(D) antibody;

FIG. 6 depicts another plasmid vector, pST4, which contains the samesequence encoding the scFv of anti-Rh(D) antibody;

FIG. 7 depicts a third plasmid vector which contains this same sequenceencoding the scFv of anti-Rh(D) antibody;

FIG. 8 depicts a plasmid vector of the pKC3B type which contains thesequence encoding multimeric CR1.

In all these figures, the restriction enzymes enabling the heterologoussequence to be inserted are indicated by their standard nomenclature.

FIG. 9 depicts the result which is obtained with multimeric scFv whenagglutinating red blood corpuscles which either do or do not exhibit therhesus antigen. The tube in A depicts the positive control, in which theO Rh+ red blood corpuscles are agglutinated by a native anti-R(h)monoclonal antibody; tube B depicts the negative control, in which ORH−0 red blood corpuscles are not agglutinated by the multimericanti-R(h) scFv antibodies; tube C depicts the assay in which O Fh+ redblood corpuscles and anti-Rh+ scFv are agglutinated; tube D is anothernegative control in which the O Rh− red blood corpuscles are mixed witha culture medium which lacks antibody.

FIG. 10 depicts the profile which is obtained in flow cytometry afterbinding an anti-Rh(D) antibody-CR1 heterochimera to erythrocytes.

Five tracks are depicted, in which:

-   -   the first track depicts non-papainated O Rh+red blood corpuscles        having a CR1 density of 180 sites;    -   the second track depicts non-papainated O Rh+ red blood        corpuscles having a CR1 density of 550 sites;    -   the third track depicts papainated O Rh+ red blood corpuscles        which have lost their density of 180 CR1 sites; these        erythrocytes, which have been reconstituted with respect to CR1        using the anti-Rh+ scFv/CR1 heterochimera, are expressing the        supraphysiological density of 1200 CR1 sites per erythroctye;    -   tracks 4 and 5 depict controls, which are papainated O Rh+ red        blood corpuscles in the case of track 4 and papainated O Rh− red        blood corpuscles treated with the anti-Rh(D) scFv /CR1        heterochimera in the case of track 5.        I—Construction of the CR1-C4BP Chains

The advantage of using CR1 in a multimeric construct according to theinvention results from the studies carried out by the inventors on thephysiological fate of CR1 in the normal subject. Thus, the inventorshave been able to determine the parameters of a physiological catabolismof erythrocytic CR1 and its relationships with the genetic polymorphismof erythrocytic CR1 density. They have also been able to clarify thecatabolism of erythrocytic CR1 in lupus patients, that is patients whoare suffering from disseminated lupus erythematosus. The distributionamong lupus patients and normal subjects of the different genotypes oflength polymorphism and CR1 C3b/C4b-binding site number polymorphism hasalso been studied. Recombinant CR1 molecules which make it possible tochange the erythrocytic CR1 density in order to restore thephysiological state of the erythrocytes or to produce “armed”erythrocytes having “supraphysiological” densities of CR1 have then beenprepared. The potential of soluble CR1 was demonstrated in differentmodels, in particular models of experimental myocardial ischemia and ofArthus phenomenon. A molecule of multimeric soluble CR1 is produced andits anti-inflammatory power, its plasma life span and its distributionspace are studied in the animal. Reduced monomers are coupled chemicallyto erythrocytes by means of their free SH group. In this way,erythrocytes are armed with supraphysiological densities of CR1, withthe CR1 being presented in a functional manner, and the ability of theerythrocytes to bind artificial C3b-opsonized Hbs antigen/anti-HBsantibody immune complexes is then studied.

The results which were obtained with an anti-Rh(D) antibody are shown inExample I below.

The constructs which were used for carrying out the C4BP-CR1transduction are depicted in FIGS. 3, 4 and 8, in plasmids pMAMneo,pCDM8 and pKC3b.

a) Construction of pMAMneo CR1-C4BP:

The complementary DNA encoding CR1 had been inserted into the Xho I andNot I sites in plasmid pCDM8 (due to the kindness of T. J. Bartow, D. T.Fearon and W. Wong, John Hopkins Hospital, Baltimore, U.S.A.). Thesequence encoding the extramembrane moiety of CR1 is extracted bydigesting this plasmid with the restriction enzymes Xho I and Bal I. TheC-terminal moiety of C4BP is amplified using the primers5′-GAGACCCCCGAAGGCTGTGA-3′, and 5′-CTCGAGTTATAGTTCTTTATCCCAAGTGG-3′,with this second primer containing a stop codon and a Xho I restrictionsite.

The sequences encoding the C-terminal moiety of C4BP and theextramembrane moiety of CR1 are inserted into pMAMNeo (Clontech, PaloAlto, USA) at the Xho I site (FIG. 3) CR1-C4BP by digestion with Xho I,and inserted into plasmid pCDM8 (Invitrogen, San Diego, USA).

b) Construction pCDM8 CR1-C4BP:

The sequence encoding the CR1-C4BP fusion protein was extracted frompMAMNeo CR1-C4BP by digestion with Xho I and inserted into plasmid pCDM8(Invitrogen, San Diego, USA) (FIG. 4).

c) Construction pKC3b CR1-C4BP:

The sequence encoding the CR1-C4BP fusion protein was extracted frompMAMNec CR1-C4BP by digestion with Xho I, and inserted into plasmidpKC3b (FIG. 8).

II—Construction of the Recombinant Multimer:

A C-terminal fragment of the α chain of C4BP was recopied from genomicDNA by means of PCR. It is found in one single exon. The minimum size isbeyond the second cysteine proceeding from the C-terminal end, with theoptimum size being a few amino acids beyond that, creating a spacer offrom 5 to 10 amino acids, that is 58 AA in all.

The maximum size selected is of 6 SCRs, in order to avoid theC3b-C4b-binding site. This maximum fragment is synthesized from a cDNAprepared from C4BP rRNA, since the fragment is made up of several exons.The optimum fragment of the C-terminal moiety of C4BP is recopied onceagain by means of PCR using primers which are provided at their endswith arms containing enzyme restriction sites which are adequate forinserting the fragment into a given vector which already contains thegene for the protein which it is desired to multimerize. An enzyme siteclose to the C-terminal moiety of this protein, or which is located inits extramembrane moiety, is selected which enables the multimerizingfragment to be inserted in the 3′ position.

The 3′ end of the multimerizirg fragment is linked either to a site inthe vector or to a site beyond in the gene for the protein of interest.That part of the gene for the protein of interest which is located 3′ ofthe multimerizing fragment is in any case no longer translated since themultimerizing fragment contains a stop codon.

It is therefore in this way possible to modify an expression vectorcontaining the gene for a given protein very readily by simply insertingthe fragment without any other alteration.

The vectors pCDM8, ST4 and pMAMneo have been used for the differentexamples of applying the multimeric system according to the invention.

The skilled person will always know how to find vectors which currentlyexist or which could be developed and which are/could be able to exhibitthe optimum efficacy for transducing the fusion protein into cells andexpressing it.

APPLICATION EXAMPLE NO. 1

Prevention of Anti-Rh(D) Alloimmunization.

Heteromultimeric molecules combining erythrocytic functions and CR1 areproduced within the context of preventing anti-Rh(D) alloimmunization.They will make it possible to bind CR1 readily to erythrocytes, therebyensuring the achievement of CR1 densities which can be fully controlled.

The antibody molecule used to generate the anti-Rh(D) scFv was producedand sequenced in Philippe ROUGER's laboratory at the Institut Nationalde Transfusion Sanguine [National Blood Transfusion Institute] (INTS)(9).

Construction of Vectors which Comprise the Sequence Encoding theAnti-Rh(D) scFv and the Terminal Moiety of the α Chain of C4BP.

An epitope site of an anti-rhesus antibody was first of all reduced downto a structure of the scFv (denoting single-chain Fv) type forexpression in E. Coli by means of transfecting with a phage vector.

Constructs of the scFv type are antibody fragments which represent thevariable moiety of the antibody and only contain one single chain. Thistechnique has been described by G. WINTER (10). The sequence encodingthis scFv was then transferred into an expression vector after addingthe multimerizing system.

We have described above the construction of expression vectors whichcarry the sequence encoding the scFv of the anti-Rh(D) antibody andwhich are depicted in FIGS. 6 and 7.

The C-terminal moiety of C4BP was amplified using the primers5′-GCGGCCGCAGAGACCCCCGAAGGCTGTG-3′, which contains a Not I restrictionsite, and 5′-CCACTTTGGATAAAGAACTATAA-3′, which contains a Xho Irestriction site.

This fragment was inserted into the Not I site 3′ of the anti-Rh(D) scFvgene. This sequence was then inserted into plasmid pCDM8 and insertedbetween the Hind III and Xho I sites of pKC3b. This construct isdepicted in FIG. 5.

FIG. 5 depicts another construct of anti-Rh(D) scFv-C4BP in plasmid pKC3B.

The plasmids are then used to transduce animal cells, in particular CHODHFR⁻ cells.

The CHO-DHFR⁻ cells (American Type Culture Collection, Rockville, USA)were transfected using the calcium phosphate technique (Calciumphosphate transfection kit, 5 prime 3 prime Inc., Boulder, U.S.A.).These cells are cultured in HAM medium lacking in Hypoxanthine andThymidine (Biochrcm, Vindelle, France) containing 10% dialysed calfserum (FCS, GIBCO BRL, Paisley, Scotland) and 1% glulamine (Sigma, St.Louis, USA).

The functionality of the reconstituted multimeric proteins, which wereeither C4BP-scFv or C4BP-Rh(D)/CR1, was studied.

Success was achieved in producing functional multimeric scFv, as wasdemonstrated (i) by biosynthetic labeling and immunoprecipitationfollowed by SDS PAGE analysis, (ii) by detecting erythrocyte-bound multianti-Rh(D) scFvs by flow cytometry, (iii) by the ability of multianti-Rh(D) scFv chimeras to agglutinate papainated red blood corpusclesat weak ionic strength, (iiii) by analyzing molecular interactions usingan instrument for detecting evanescent waves (IASys FISONS), with theinstrument verifying the binding of the multimeric anti-Rh(D) anti scFvchimera to erythrocytes.

Subsequently, the feasibility of creating heteromultimers which combinedifferent functions was established by preparing an anti-rhesus Dantibody/CR1 chimera. Its ability to function was demonstrated by flowcytometry. The results which were obtained by flow cytometry aredepicted in FIG. 10. It is clear from this figure that, if tracks 3 and5, in which the red blood corpuscles are Rh+ and Rh−, respectively, arecompared, it is observed that only the red blood corpuscles whichpossess the surface antigen are agglutinated. This figure also showsthat this agglutination is not due to the effect of the papain since thepapainated Rh+ red blood corpuscles are not agglutinated by CR1.

It was therefore possible to bind a supraphysiological density of CR1molecules on erythrocytes which had been previously papainated, andtherefore depleted of CR1, by binding a mixed multimeric anti-Rh(D)antibody/CR1 chimera to he rhesus D molecules.

APPLICATION EXAMPLE NO. 2 Extracellular Destruction of HIV

Even though the humoral immune response is unable to eradicate the HIVvirus, it is nevertheless effective against a large number of infectiousagents against which neutralizing antibodies are regularly produced byvaccinated subjects.

Natural antibodies can confer protection against a large number ofbacterial or viral infectious agents which infect other species.

Certain antigenic motifs have been characterized as targets for thistype of antibody in connection with their role in the peracute vascularrejection of xenogeneic transplants. The potentially unfavorable role ofthe humoral immune response and of complement activation with regard toinfection with HIV virus has been demonstrated.

Under certain circumstances, opsonization of the virions can facilitateingestion by macrophages or binding of the virions to lymphocytes by wayof cell receptors for complement or the IgG Fc fragment. On the otherhand, the HIV virus, as an enveloped virus, is extremely sensitive tothe lytic action of complement when the latter has been sufficientlyactivated to initiate its final lytic path and binding of its membraneattack complex. Xenogenic retroviruses are destroyed extremelyefficiently by normal human serum, due to the existence of naturalantibodies, by way of activating complement. In the 1970s, thisphenomenon had even led to the conclusion that the human species wasnaturally immune to retroviruses.

The sought-after aim is to reroute a lytic huoral immune response towardHIV virions, with attachment to the virions being effected by the CD4component of a recombinant heteromultimeric protein. For this, theinventors have taken into account the fact that while the HIV virusexhibits a very high degree of variability, this variability isnevertheless limited by the necessity of having constantly to preservean ability to bind to CD4, which ability is required for the virus topenetrate the cell and is essential for the virus. In a reciprocalmanner, the CD4 molecule is able to bind to all HIV virions, in contrastto a large number of neutralizing antibodies, which have a spectrum ofefficacy which is restricted to a small number of subtypes. The solubleCD4 molecule inhibits cell infection by HIV. However, the concentrationswhich are required for it to have this effect, particularly forneutralizing wild-type isolates, render its clinical use impracticable.Various attempts have been made to improve its half-life and itsavidity, and/or supply an Fc gamma function which effects cell bindingor complement activation using constructs of the bivalent or tetravalentCD4/IgG type.

The inventors have developed a heptameric multivalent CD4 molecule usinga multimerizing C4BP system whose biological efficacy in vitro wasdemonstrated by inhibiting infection of susceptible cells by HIV atinhibitory concentrations which are common for monomeric soluble CD4.

In the present example, rather than inhibiting cell penetration by thevirus, the inventors have sought to destroy the virus extracellularlyusing soluble molecules which are able, on the one hand, to bind to thevirion and, on the other, to supply an antigenic effector function whichelicits destruction of the virus by means of an antibody response whichdepends on complement which pre-exists in the individual patient. Thisresponse is directed against an antigen which has nothing to do with theHIV but in relation to which the immune system has nevertheless beendemonstrated to exhibit neutralizing and lytic efficacy. In other words,since the HIV virus has the ability to “disguise” itself, to “hide”itself or to “raise decoys”, the inventors have sought to embellish thevirus with targets which the immune system of the individual patientknows how to identify and deal with efficiently.

In order to do this, two plasmid constructs were made which respectivelycarry CD4 or the fragment of CD4 which carries the “ligand” fraction andan antigen.

a) Construction of Vector ST4 CD4-C4BP

The last 183 nucleotides of the sequence encoding C4BP were amplified bymeans of PCR, on genomic DNA, using the following primers:5′-GAGACCCCCGAAGGCTGTGTGA-3′ and5′-ATTTCTAGAGAGTTATAGTTCTTTATCCAAAGTGGA-3′, with this latter primercontaining a stop codon and a restriction site for Xba I. This PCRfragment was linked at its 5′ end to the following double-strandedsynthetic oligonucleotide sequence:

5′ CCGGGACAGGTCCTGCTGGAATCCAACATCAAGGTTCTGCCCACAG- 3′.

This fragment encoding the C-terminal end of the extramembrane moiety ofCD4 and having an Ava I site at its 5′ end. This sequence was insertedinto the Ava I and Xba I sites in plasmid sT4 CD4, containing theconstruct encoding soluble CD4, and this construct is depicted in FIG.1.

b) Construction of C4BP-antigen Fusion Molecules

It is firstly a matter of trying to determine which parameters directthe anti-gp 120 antibody response of infected subjects to complementactivation up to the C3 amplification loop, resulting in opsonizationwhich is relatively advantageous for the individual patient, without,for all that, being accompanied by activation of the final commonpathway which would result in lysis of the virion. The role of surfacemolecules which inhibit activation of complement and which the virionhas taken from the cell surface has been demonstrated. The shedding ofenvelope particles when antibody is being bound also militates againstterminal activation of complement. Activation of the final commoncomplement pathway requires the C3 to have a density which is criticalfor activation in order to initiate C5 conversion. This conversion isnot brought about by IgG being bound to gp 120 epitopes which are toodistant from each other.

The use of the constructs of the invention to supply a “cluster” ofantigens for each binding site on the virion thus makes it possible totrigger adequate local activation of complement.

Various categories of antigen have been considered: vaccinatingantigens, bacterial antigens against which humans are universallyimmunized, and xenogenic antigens which are the targets of naturalantibodies.

-   -   vaccinating antigens which exist in the form of cloned genes and        which encode a protein which can be expressed in eukaryotic        cells (Hbs antigen, tetanus toxoid, etc.),    -   bacterial antigens to which there exists strong immunity in        humans (Escherichia coli, Klebsiella, Shigella flagellin or        Salmonella antigen),    -   molecules which possess protein sequences which accept xenogenic        glycosylations may also be envisaged after they have been        produced by animal cells which possess strong glycosyl        transferase activities which will attach to the proteins        carbohydrates which are the targets of natural antibodies and        which are known to react strongly in xenotransplants (for        example: the alpha-galactosyl group, which is an impediment to        xenogeneic pig/man transplants).

These miniantibodies will be used as agents for binding heterochimerasto erythrocytes, of which chimeras they only represent one valency ofthe C4BP β type, which valency is linked to a multimeric antigenicmolecule of the heptameric C4BP α type. The most effective antigenicsystem will therefore be selected in a readily quantifiable screeningtest. It will then be transferred into a recombinant CD4/antigen targetchimera whose different CD4/antigen ratios (1CD4/7 antigens or nCD4/Mantigens) will be tested in an in-vitro model of the inhibition of viralinfection.

These miniantibodies will be used as agents for binding heterochimerasto erythrocytes, of which chimeras they only represent one valency ofthe C4BP beta type, which valency is linked to a multimeric antigenicmolecule of the heptameric C4BP alpha type. The most effective antigenicsystem will therefore be selected in a readily quantifiable screeningtest. It will then be transferred into a recombinant CD4/antigen targetchimera whose different CD4/antigen ratios (1CD4/7 antigens or nCD4/Mantigens) will be tested in an in-vitro model of the inhibition of viralinfection.

The most interesting target antigens were inserted into heteromultimericconstructs containing CD4 and tested for their ability to enable HIVvirions to be destroyed in the presence of human serum and complement,with residual infectivity being evaluated in an in-vitro test of theinhibition of cell infection.

Materials and Methods:

In addition to the abovementioned constructs, the techniques employedfor transfecting and culturing cells were as follows:

Transfection:

DHFR⁻ CHO cells (American Type Culture Collection, Rockville, USA) weretransfected using the calcium phosphate technique (Calcium phosphatetransfection kit, 5 prime 3 prime Inc., Boulder, U.S.A.).

Cell Culture:

The cells are cultured in HAM medium lacking Hypoxanthine and Thymidine(Biochrom, Vindelle, France) but which contains 10% dialyzed calf serum(FCS GIBCO BRL, Paisley, Scotland) and 1% glutamine (Sigma, St. Louis,USA).

The cells which have been transfected with pMAMNeo are selected on thebasis of their ability to resist neomycin (G418, 0.7 microg/ml) (Sigma).Dexamethasone (0.8 microg/ml) is used to induce the production of mCR1in the cells which are transfected with the pMAMneo CR1-C4BP.

In their experiments, the inventors used an appliance for culturingcells continuously in hollow fibers in order to produce recombinantproteins on the scale of a few milligrams, or a few tens of milligrams,of recombinant proteins. Most of the experiments can be carried outusing crude or concentrated culture supernatants. Small-scale purifiedpreparations have also been prepared.

The oligonucleotide syntheses which were employed for constructing thevectors were carried out in order to fit the C-terminal C4BP fragment toeach construct. The nucleotide sequences were also determined on anautomated fluorescence sequencer in order to check the constructs.

Comments

The multimeric proteins of the invention, and their use in producing amedicament for prophylactic or therapeutic purposes, or their use as adiagnostic or research tool, are very powerful.

Using them can be an efficient tool for analyzing physiologicalmechanisms in the immune response as well as for understanding thephysiopathology of certain disorders of the immune system.

These molecules should make it possible to intervene in the immunesystem in a more sophisticated manner, thereby opening up thepossibility of being better able to study large numbers ofphysiopathological mechanisms in vitro. In certain cases, thisimmunointervention will open up the route to manipulating the immunesystem in vivo for therapeutic purposes. The molecules are, therefore,at one and the same time tools for carrying out clinicalphysiopathological research and in vitro experimental research and alsotherapeutic tools for use in vivo.

BIBLIOGRAPHY

-   1. Matsuguchi T., Okamura S., Aso T., Niho Y., Molecular cloning of    the cDNA coding for PRP: identity of PRP as C4BP. Biochem Biophys    Res Commun 1989; 1: 139-44.-   2. Scharfstein J., Ferreira A., Gigli I., Nussenzweig V., Human    C4-binding protein. I. Isolation and characterization. J. exp Med    1978; 148: 207-22.-   3. Fujita T., Gigli I., Nussenzweig V., Human C4-binding protein II.    Role in proteolysis of C4b by C3b-inactivator. J. Exp Med 1978; 148:    1044-51.-   4. Chung L. P., Bentley D. R., Reid K. B. M., Molecular cloning and    characterization of the cDNA coding for C4b-binding protein, a    regulatory protein of the classical pathway of the human comlement    system. Biochemistry 1985: 230: 133-41.-   5. Monte G. Thrombosis and Haemostasis—69(1)86(1993).-   6. Hillarp A., (1990) PNAS vol. 87 pp. 1183-1187.-   7. Hillarp A., (1991) Scand. J. Chin. Lab. Invest.

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-   8. Fanger M. W., Immunomethods 1994-p. 72 to 81 “Production and use    of anti-FcR bi-specific antibodies”.-   9. Goossens D., Champomier F., Rouger Ph. and Salmon Ch., Human    monoclonal antibodies against blood group antigens: preparation of a    series of stable EBV Immortalized B clones producing high levels of    antibody of different isotypes and specifites. J. of immonological    methods., 101, 193, 1987.-   10. Winter G., Nature 1990-348-p. 552-554. M. Cafferty J., Griffiths    A., Winter G., Chiswell. “Phage antibodies filamentous phase    displaying antibody variable domains”.

1. A recombinant multimeric protein, comprising a) a polypeptide fusionmonomer A, which consists of a C-terminal fragment of the α chain ofC4BP consisting of SEQ ID NO: 11 and containing one or two cysteineresidues, and a polypeptide fragment which is heterologous in relationto said α chain, b) a polypeptide fusion monomer B, which consists of aC-terminal fragment of the β chain of C4BP consisting of SEQ ID NO: 12and containing one or two cysteine residues, and a polypeptide fragmentwhich is heterologous in relation to the β chain, monomer A and monomerB being linked to each other by a disulfide bridge between a cysteine ofthe α chain C terminal fragment and a cysteine of the β chain C-terminalfragment to form said multimeric protein.
 2. The recombinant multimericprotein according to claim 1, wherein the ratio of the number ofmonomers A/B varies between 7/1 and 5/3.
 3. The recombinant proteinaccording to claim 1, wherein the heterologous fragments in monomer Aand in monomer B are ligands of the immune system, selected from groupconsisting of CD lymphocyte surface proteins, antibodies, antibodyfragments, antigens and antigen fragments.
 4. The recombinant multimericprotein according to claim 3, wherein the antibodies or antibodyfragments are specific for anti-Rh (D).
 5. The recombinant multimericprotein according to claim 1, wherein the polypeptide fusion monomer Acomprises a ligand selected from the group consisting of an antigen, atherapeutic enzyme, a CD35, CR1, and an antibody.
 6. A compositioncomprising a recombinant multimeric protein according to claim
 1. 7. Therecombinant multimeric protein according to claim 1, wherein thecysteine residues of the C-terminal of the α chain and the C-terminalfragment of the β chain each include two cysteine residues.
 8. Therecombinant multimeric protein according to claim 7, wherein thecysteine residues of the C-terminal of the α chain are located atpositions 498 and 510 of SEQ ID NO: 7 and the cysteine residues of theC-terminal of the β chain are located at positions 510 and 549 of SEQ IDNO:
 8. 9. The recombinant multimeric protein according to claim 1,comprising at least one each of monomer A and monomer B, and at leastseven monomers A and B in all.
 10. The recombinant multimeric protein,comprising a) a polypeptide fusion monomer A, which consists of aC-terminal fragment of the α chain of C4BP consisting of SEQ ID NO: 9,and b) a polypeptide fusion monomer B, which consists of a C-terminalfragment of the β chain of C4BP consisting of SEQ ID NO:
 10. 11. Arecombinant multimeric protein, comprising a) a polypeptide fusionmonomer A, which consists of a cysteine-containing C-terminal fragmentof the α chain of C4BP consisting of SEQ ID NO: 11 and containing one ortwo cysteine residues, and a polypeptide fragment which is heterologousin relation to said α chain and is a ligand of the immune system, b) apolypeptide fusion monomer B, which consists of a cysteine-containingC-terminal fragment of the β chain of C4BP consisting of SEQ ID NO: 12and containing one or two cysteine residues, and a polypeptide fragmentwhich is heterologous in relation to the β chain and is a ligand of theimmune system, monomer A and monomer B being linked to each other by adisulfide bridge between a cysteine of the α chain C terminal fragmentand a cysteine of the β chain C-terminal fragment to form saidmultimeric protein, wherein said recombinant multimeric proteinactivates complement to induce opsonization of cells.